Refractory anemia with ringed sideroblasts and thrombocytosis without JAK2 V617F mutation: report of three cases.

In the WHO classification, there is a provisional entity called Myelodysplastic/Myeloproliferative Neoplasm, Unclassifiable (MDS/MPN, U). Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T) was included in this category. Recently published studies report a small percentage of patients with RARS-T. Sixty percent of these have JAK2 V617F mutation, which can suggest the coexistence of two pathological conditions (MDS and MPN). In this paper, we analyzed three patients diagnosed with RARS-T in the Department of Hematology, "Fundeni" Clinical Institute, Bucharest, Romania, during the period 2005-2011. The patients were investigated with cytogenetic exam and molecular biology. In these three cases were identified morphological features of multilineage dysplasia (two-lineage dysplasia in two cases and three-lineage dysplasia in one case). In two cases, thrombocytosis was under 1000×10(3)/µL and clinical evolution was similar to the myelodysplastic syndrome (transfusion dependent anemia with response to administration of erythropoietin). In the third case, the platelets were over 1000×10(3)/µL and with response to the treatment with Hydrea, which improved anemia. JAK2 V617F mutation was not identified in any case. RARS-T remains a provisional entity and requires a complex investigation of patients for the correct diagnosis of these patients. Therapeutic options should be personalized to each case in part because there is not yet a standardized treatment of these patients.

Prenatal molecular diagnosis of beta-thalassemia: report on the first two cases in Romania.

Thalassaemia major is a classical example of a disease that can be prevented by prenatal diagnosis. In Romania there are currently 300 patients with thalassaemia major under the management of specialized institutions. Prenatal diagnoses of thalassemia have offered a new dimension to the prevention of this disease, but in order to implement prenatal diagnosis, knowledge of mutations and of their incidence is essential. Molecular testing using Denaturing Gradient Gel Electrophoresis (DGGE) scanning and direct mutation detection with Amplificaton Refractory Mutation System-PCR (ARMS-PCR) and Restriction endonuclease Analysis of PCR fragments (PCR-RFLP) was performed by using amplified DNA from amniotic cells samples, while mutations in the parents were determined in advance. Using our experience in molecular diagnosis, we were able to perform the first prenatal diagnosis for two young couples at risk for thalassaemia major. Foetal samplings were collected by amniocentesis and chorionic villus sampling in the second trimester of the pregnancies. Maternal contamination of the foetal DNA was ruled out by STR genotyping. The prenatal diagnosis revealed affected foetuses with homozygous status of beta-thalassemia major. The IVSI-110 (G-A)/IVS II-745 (C-G) genotype in the first case foetus and ed 8 (-AA)/cd 8 (-AA) in the second case foetus were reported. The results of this study point to a successful future prenatal diagnosis of beta-thalassnemia in Romania, using a rapid and accurate molecular method. Together with the implementation of proper preventive health measures and the education of parents regarding their carrier status, we are hoping that this method will be used as the common application approach to decrease the incidence of thalassacmia major.

Total body irradiation prior to bone marrow transplantation--the experience of the Institute of Oncology Prof. Dr. Al. Trestioreanu Bucharest.

PURPOSE: To present the technique of total body irradiation (TBI), applied for the first time in Romania, at the Institute of Oncology Bucharest, as part of stem cell transplantation for hematological malignancies. PATIENTS AND METHODS: The total dose administered was 12 Gy at the reference point, 2 Gy/fraction, one fraction per day, 6 consecutive days, with a total dose of 8 - 11.4 Gy delivered to the lung, using Mevatron Primus linear accelerator (6 MV & 15 MV, 200-300 cGy/min in isocenter), in vivo dosimetry detectors and equipment for the reference dosimetry, personalized blocks for lung shielding sustained by polymethylmethaacrylate (PPMA) plate, Simulix HP simulator, and computer tomographic (CT) scans. Techniques used were: a) two parallel opposed anteroposterior / posteroanterior (AP/PA) fields with the patient in prone and supine position; b) two parallel opposed lateral fields with the patient placed on a lateral table, at 320 cm from the source. The percentage depth dose, tissue maximum ratio (TMR), off axis ratio (OAR) and the reference dose rate were measured for every patient's geometrical characteristics, with an uncertainty of +/- 2.2% and were used to calculate monitor units and to evaluate the dose in organs at risk (lungs, gonads, eyes etc). RESULTS: 5 patients (3 with the AP/PA technique and 2 with the lateral technique) were irradiated. All patients completed their irradiation in good clinical condition. The acute side effects were minimal (WHO grade 1: nausea/ vomiting--all patients; diarrhea--1 patient; headache--2 patients; photophobia and diplopia--1 patient; head and neck skin erythema--all patients). Because of the short follow-up period no safe evaluation of late side effects can be done. However, during this period one patient developed a non-aggressive form of chronic liver graft vs. host disease (GVHD) and one patient died due to acute GVHD. CONCLUSION: TBI as part of stem cell transplantation for hematological malignancies was successfully realized at our Institute, with favorable clinical results. This technique is easy to carry out and reproducible.

Scanning of beta-globin gene for identification of beta-thalassemia mutation in Romanian population.

Beta-thalassemia is uncommon (0.5%) in the Romanian population, but it must be considered in the differential diagnosis of hypochromic anemia. The molecular characterization of beta-thalassemia is absolutely necessary for molecular diagnosis, as well as any genetic epidemiological study in this region. Molecular analyses consist of mutation detection by molecular scanning of beta-globin gene. This gene has 3 exons and 2 introns, involved in beta-thalassemic pathogenesis. Clinical application of DNA analysis on beta-thalassemic chromosomes allowed characterization of 29 persons with different beta-thalassemia mutations among 58 patients with anemia. The experimental strategy was based on sequential PCR amplification of most of the beta-globin gene and running on denaturing gradient gel electrophoresis of amplification products. Definitive characterization of mutations in samples identified with shifted DGGE patterns was performed ARMS-PCR and/or PCR-restriction enzyme analysis methods. Eight different beta-thalassemia alleles were identified, the most common being IVS I-110 (G-A) and cd 39 (C-T). Comparison of overall frequency of mutations in the neighboring countries, shows that these results are in the frame of overall distribution of these mutations in Mediterranean area, especially in Greece and in Bulgaria. Molecular diagnosis is useful for differentiating mild from severe alleles, for genetic counseling, as well as for mutation definition in carriers, identified by hematological analysis necessary for prenatal testing and genetic counseling.

[A case of disseminated intravascular coagulation--a paraneoplastic manifestation of a borderline ovarian tumor]. / Consideratii asupra unui caz de coagulare intravasculara diseminata (CID)--manifestare paraneoplazica a unei tumori ovariene de granita.

A 24-year-old-woman was admitted because of disseminated intravascular coagulation (DIC), menometrorrhagia and galactorrhea. The investigations performed showed a right adnexal tumor after the equilibration of DIC with plasma substitution, we performed a right adnexectomy with limited excision of peritoneal. The pathologic examination showed a focus of endometriosis on the right ovary who had a polycystic look and a right adnexal fibromyxoma (premalignant lesion). The post operative evolution was good, with the loss of entire onco-hemato-endocrinologic picture. We describe the interrelation between DIC (paraneoplastic syndrome), menometrorrhagia and galactorrhea, the pathologic hypothesis and the treatment of DIC.

The value of the ultrastructural pattern of myeloperoxidase (MPO), platelet peroxidase (PPO) and acid phosphatase in identification of blastic cell types during acute transformation of chronic myeloproliferative disorders (cMPD).

We established the cell line of bone marrow and blood cells provided by 19 cases of cMPD by ultrastructural studies of their enzymatic content. Myeloblasts, megakaryoblasts and lymphoblasts were identified by the presence of myeloperoxidase, platelet peroxidase and acid phosphatase, respectively.

Autoimmune hemolytic anemia with warm antibodies complicated with an intercurrent attack of hemolysis with cold agglutinins in a case of Waldenström disease.

The paper presents a case of autoimmune hemolytic anemia (AIHA) with warm IgG antibodies associated with Waldenström's disease in which in the stage of compensated hemolysis after treatment, there appeared a severe hemolytic attack induced by transitory cold agglutinins with high thermal amplitude. The case described is a novelty by the intrication of two autoantibody populations which causes autoimmune hemolysis. The difference from other similar associations is discussed.

Ultrastructural aspects of megakaryocyte/platelet system in essential thrombocythemia.

Six cases of essential thrombocythemia (ET) have been investigated in bone marrow as well as in blood cells. In majority of the cases, the bone marrow aspirate was hypercellular and presented an increased number of megakaryocytes, some of them with displastic appearance. The ultrastructural pattern of platelet peroxidase permitted us to identify atypic megakaryocytes and megakaryoblasts, particularly when present in pheripheral blood.

A complex translocation t(1-12-11) in a patient with hodgkins-disease.

A new complex translocation t(1;12;11) (p32;q24;q23) was found as the sole abnormality in spleen cells from a patient with lymphocyte predominant subtype of Hodgkin's disease. The patient is in complete remission 4.5 years after diagnosis.

Introduction to the study of adult T-cell leukemia and HTLV-I sero-positivity in Romania.

The study presents 6 cases of leukemia/lymphoma (with mature T-cells) corresponding to the diagnostic criteria of adult T-cell leukemia (ATL): adult age onset of leukemia/lymphoma, organomegaly but normal mediastinum, leukemic cells with typical morphology and phenotype hypercalcemia. The evolution of disease was severe of subacute with resistance or partial response to therapy. The virologic assays were positive according to the ELISA test in four cases of which two presented, according to the Western blot assay, HTLV-I infection. The epidemic aspect of this infection is discussed as well as the possibility of contamination and the geographic spread of the places of origin of the patients. Emphasis is laid on the young age (22-26 years) of three of the patients infected, as a peculiar feature of the disease in Romania.

Ultrastructural features in an erythrophagocytic T4 lymphoma.

Electron microscopic investigation allowed the identification and characterization of the atypical malignant cells in a case of T-cell lymphoma. The proliferating cells were endowed with characteristics belonging to two different lineages: phagocytic ability, complement receptors and ultrastructural features proper to the macrophagic lineage, and T-cell determinants (E receptors, T3, T4 and T11 antigens). The cells were peroxidase and esterase negative. The erythrocytes were partially or completely dehemoglobinized and presented the phenomenon of autolysis in different stages of evolution. Because this lymphoma is difficult to diagnose and is apparently resistant to therapy, its recognition and further study are warranted.